Over-expressing homologous antigen vaccine and a method of making the same

ABSTRACT

This invention relates to an over-expressing homologous antigen vaccine, a method of producing the same, and use of the vaccine for prophylaxis or treatment of vertebrates at risk of or suffering from disease caused by a pathogenic micro-organism. The vaccine is an attenuated or avirulent pathogenic micro-organism that over-expresses at least one homologous antigen encoded by at least one gene derived from the pathogenic micro-organism, and may also express a heterologous antigen.

The invention described herein was made under a grant from the UnitedStates Department of Agriculture. Therefore, the U.S. government mayhave certain rights in this invention.

The invention pertains to an over-expressing homologous antigen vaccine,a method of producing the same, and a method of using the vaccine forprophylaxis or treatment of a vertebrate suffering from or at risk froma pathogen. The vaccine is derived from an attenuated or avirulentversion of the pathogen, and over-expresses one or more genes from thepathogen, thereby providing immunity greater than that induced by avaccine of the same pathogen without over-expression of a gene.

BACKGROUND OF THE INVENTION

Vaccines are used to protect against diseases, which are caused bypathogens. These pathogens are microbial organisms, such as bacteria andviruses, which affect animals, including humans. Vaccines are primarilyderived from a pathogen by producing and administering either: a) anattenuated or avirulent version of the pathogen; b) the killed pathogen;c) extracted protective antigens or antigen mixes of the pathogen(homologous antigens); or d) a micro-organism expressing one or moreprotective antigens encoded by cloned genes originating in a microbialpathogen different from the vaccine strain (heterologous antigens).

Vaccines for both bacteria and viruses are engineered frommicroorganisms expressing one or more protective antigens, as describedby K. Jones and M. Sheppard in Designer Vaccines, CRC Press (1997).Vaccines are intended to produce an immune response in the recipientconsisting of at least one of an antibody mediated or T cell mediatedimmune response, thereby preventing future infection by a pathogen, orfighting a current pathogenic infection. In particular, vaccines againstfacultative intracellular pathogens, those growing inside the cells ofthe infected host, need to induce a strong and appropriate cell mediatedimmune response. In contrast, vaccines against obligate extracellularpathogens need to induce an appropriate antibody mediated immuneresponse. Often, regardless of the pathogen, an appropriate combinedantibody and cellular mediated immune response leads to sufficientprotection or relief from infection. In order to achieve this protectionor relief from infection, vaccines may express one or more homologousantigens, heterologous antigens, or a combination of both.

Vaccines may be administered to vertebrates both to prevent and treatinfection by pathogens. Thus, vaccines are frequently administered toprevent the spread of a disease caused by a pathogen. In particular,herd animals, such as cows, goats, sheep and swine, are often vaccinatedto prevent the spread of a disease among members of the herd. Further,because certain diseases may travel between vertebrates, includingtravel between various animals and between animals and humans, vaccinesare used to prevent the spread of disease between various species,usually by administration to the infected animal and other uninfectedanimals in the immediate vicinity. However, other animals in the areawhich are less likely to contract the disease may also be vaccinated asa prophylactic measure. For example, an infected cow and its as yetuninfected herd may be vaccinated to treat a disease and prevent itsfurther spread. As a prophylactic measure, other animals which arelikely to contract the disease from the infected cow, such asneighboring cows, sheep or humans, may be vaccinated as well.

It has been found that vaccines derived from an attenuated or avirulentversion of a pathogen are highly effective in preventing or fightingdisease caused by that pathogen. In particular, it is known that suchattenuated or avirulent pathogens can be modified to expressheterologous antigens (antigens which are derived from a pathogen of adifferent species). In order to express heterologous antigens in adesired attenuated or avirulent pathogen, a gene encoding an antigencapable of providing protection against the pathogen is identified fromthe deoxyribonucleic acid of a heterologous species. The desired gene isisolated and then inserted into a plasmid capable of replication andexpression in the attenuated or avirulent pathogen. The plasmid is thenintroduced into the attenuated or avirulent pathogen, and causesexpression of the heterologous antigen upon administration to a subjectvertebrate.

An example of such expression of an heterologous antigen is thebacterial vaccine Salmonella, which expresses a Streptococcus spaAprotein. See U.S. Pat. No. 4,888,170. This vaccine comprises anavirulent derivative of a pathogenic microbe of the genus Salmonella,which in turn expresses a recombinant gene derived from a pathogen ofthe species Streptococcus mutans, thereby producing an antigen capableof inducing an immune response in a vertebrate against the pathogen.

A further example of heterologous expression is Vibrio cholera vaccines.A number of live attenuated strains of Vibrio cholera have beendeveloped to vaccinate humans against cholera. See Kaper, J. B., et al.,New and improved vaccines against cholera in New Generation Vaccines(eds. M M Levin et al.) Marcel Deker, Inc., NY, 1997. Some of thesestrains over-express heterologous antigens. See Butterton, J. R. and S.B. Calderwood, Attenuated Vibrio cholera as a live vector for expressionof foreign antigens in New Generation Vaccines (eds. M M Levin et al.)Marcel Deker, Inc., NY, 1997. The immunity induced by the attenuatedvaccine strains is the result of inducing antibodies which have eitherantibacterial and/or antitoxic activities. Some strains have beenattenuated by the deletion of a number of genes encoding toxigeniccomponents, including the A subunit of the cholera toxin encoded by thectxA gene. However, in order for a cholera vaccine strain to be fullyprotective, it is necessary that the ctxB gene encoding the B subunit(to which the A subunit binds) be expressed to allow for the productionof antibodies that neutralize the cholera toxin. The ctxB gene has beenover-expressed in Vibrio cholera for the purpose of producing largeamounts of the antigen cholera toxin B (CTB). The over-expressed antigenCTB is collected, purified and used as a subunit vaccine which is theextracted CTB antigen. See Lebens M., et al., 1993, Biotechnology (NY)December; 11:1574-1578. However, although an over-expressed antigen hasitself been used as a vaccine, an attenuated or avirulent pathogen ofVibrio cholera which over-expresses the ctxB gene, or any otherhomologous gene, has not been used as a live vaccine.

Another example of heterologous expression is in Mycobacterium spp.vaccines, used to prevent tuberculosis in humans. The Mycobacteriumtuberculosis GroEL protein induces protective immunity when expressed bythe groEL gene transfected into macrophages (Silva, C. L. and Lowrie, D.B., 1994, Immunology 84:244-248), indicating that GroEL protein is aprotective antigen if presented to T cells by this type of antigenpresenting cell (APC). Naked DNA vaccines using Mycobacterium genescoding for a variety of antigens (hsp70, 85kDa, 65kDa, 36kDa, 6kDa) arealso able to induce protective immunity. See Lowrie, D. B. et al., 1997,Vaccine 15:834-838; Tascon, E. et al., 1996, Nat. Med. 2:888-892; andLozes, E. et al., 1997, Vaccine 15:880-833. It is believed that thenaked DNA vaccines work because they transfect APCs (Chattergon, M. etal., 1997, FASEB J. 11:753-763.) which in turn present the antigenappropriately to T cells, thereby inducing a protective cell mediatedimmunity. M. bovis BCG, a live, attenuated strain of Mycobacterium, isused to induce protective immunity against M. tuberculosis infection inhumans. Fine, P M. 1988, Br. Med. Bull. 44:91.

Antigen vaccines developed against Brucellosis provide examples ofhomologous antigen expression, wherein the antigen is derived from thesame species as the attenuated pathogen. Brucellosis is an infectiousbacterial disease which can be transmitted to human beings by animals.It is caused by any of a variety of species of pathogenic aerobicbacteria of the genus Brucella. In animals, Brucellosis can result inabortion and infertility. In humans, it causes fever, malaise andheadaches. This disease has been extensively studied, resulting in thedevelopment of numerous vaccines.

It is known that existing vaccine strains of Brucella, such as B.abortus strains 19 and RB51, and B. melitensis strain REV1, can bothprotect against the Brucella species from which they were derived andcross protect against infection by other species, such as B. abortus, B.melitensis, B. ovis, B. suis, B. canis and B. neotomae. See Winter, A.J. et al., 1996, Am. J. Vet. Res., 57:677; P. Nicoletti in AnimalBrucellosis, CRC Press (1990), pp. 284-296; J. M. Blasco in AnimalBrucellosis, CRC Press (1990), pp. 368-370; and G. C. Alton in AnimalBrucellosis, CRC Press (1990), pp. 395-400. New B. melitensis strainVTRM1 and B. suis strain VTRS1 also cross protect against variousBrucella species. See Winter, A. J. et al., Am. J. Vet. Res., 57:677.

In the past, one of the most commonly used vaccines to prevent bovineBrucellosis was B. abortus strain 19, as described by P. Nicoletti inAnimal Brucellosis, CRC Press (1990), pages 284-296. This particularstrain of B. abortus provided immunity in cattle with a range ofprotection from 65 to 75% depending upon a number of variables, such asthe age of the cattle at vaccination, the dose administered, the routeof administration and prevalence of Brucellosis in the vaccinated herd.

B. Abortus strain RB51, a new attenuated live Brucella vaccine (marketedas RB-51®), is a stable vaccine approved for use in the United States.See Schurig, G. G. et al, 1991, Vet. Microbiol. 26:359; and Colby, L.,1997, M.Sc. Thesis, Virginia Tech, Blacksburg, Va. Attenuation of strainRB51 is indicated by studies carried out in mice, goats and cattle. SeeSchurig, G. G., 1991, Vet. Microbiol. 28:171; Palmer R. M. et al., 1997,Am.J. Vet Res. 58:472; Roop, R. M. et al., 1995, Res. Vet. Science,51:359; and Zambrano, A. J. et al., 1995, Archivos de MedicinaVeterinaria XXVIII, No. extraordinario:119-121. In comparison to theprotection provided by strain 19, strain RB51 has been shown in singlevaccination protocols to be similarly protective in cattle. SeeCheville, N. F. et al., 1993, Amer. J. Vet Research 53:1881; andCheville, N. F. et al., 1996, Amer. J. Vet Research, 57:1153. Further,oral administration of strain RB51 in mice and cattle has indicatedprotective immunity. See Stevens, M. G. et al., 1996, Infect. Immun.64:534. In particular, the mouse model indicates that the protectiveimmunity to Brucellosis induced by strain RB51 is solely T cell mediatedbecause a passive transfer of RB51-induced antibodies does not protectagainst the disease, whereas adoptive T cell transfer does. See Bagchi,T., 1990, M.Sc. Thesis, Virginia Tech, Blacksburg, Va.; JimenezdeBagues, M. P. et al., 1994, Infect. Immun. 62:4990. It is believedthat vaccination with RB-51® confers protection by inducing productionof interferon gamma able to activate macrophages and specific cytotoxicT cells in the subject which are able to kill Brucella infectedmacrophages.

Although RB-51®, derived from B. abortus strain 2308, is the bestcurrent vaccine against Brucellosis in animals, it is still not 100%effective. None of the current Brucellosis vaccines are totallyeffective. Therefore, research continues on promising strains, such asB. abortus strain RB51. For example, expression of heterologous antigensby B. abortus strain RB51 has been described by S. Cravero, et al. 1995,Proceedings 4th Intl. Vet. Immunol. Symposium, July, Davis, Ca.,Abstract # 276; and S. Cravero et al., 1996, Conference of ResearchWorkers in Animal Diseases, Nov., Chicago, Abstract # 150.Over-expression of a homologous antigen by Brucella has been describedas a research tool for the purpose of complementing specific deletionmutants for the study of HtrA protein in B. abortus (P. H. Elzer, Inf.Immun., 1994, 62:4131), and for the study of physiological functions asdiscussed by R. Wright at an Oral Presentation of the Brucella ResearchConference on Nov. 9, 1997 in Chicago, Ill.

However, over-expression of homologous antigens of Brucella or otherpathogens, with or without concomitant expression of a heterologousantigen, has not been studied for use in vaccines. Over-expression ofhomologous antigens previously has been used primarily as a researchtool, as described above. An attenuated or avirulent pathogen modifiedto over-express an homologous antigen has not been used as a livevaccine. However, we have found that a vaccine which is an attenuated oravirulent pathogen which over-expresses one or more homologous antigens,as described herein, will provide greater protection against apathogenic disease than vaccines of attenuated pathogens which expresswild type levels of the same homologous antigens.

Therefore, the invention is directed to a vaccine, a means of producingthe vaccine, and its use for prophylaxis and treatment of a pathogenicdisease wherein the vaccine is an attenuated or avirulent pathogen whichover-expresses at least one homologous antigen, thereby providinggreater protection against and treatment of the disease caused by theunattenuated pathogen in the subject vertebrate.

SUMMARY OF THE INVENTION

The invention is directed to a live vaccine which is an attenuated oravirulent pathogen which over-expresses one or more homologous antigensof a pathogen, a method of producing the same, and a method of treatinganimals, including humans, with the vaccine. This vaccine increases thelevel of protection against the unattenuated pathogen in comparison tovaccines of attenuated pathogens expressing wild type levels ofhomologous antigens of the pathogen. In this manner, the over-expressinghomologous antigen vaccine will induce a strong cellular mediated immuneresponse and/or a strong humoral antibody response against theunattenuated pathogen in the vaccinated subject.

In particular, it is the purpose of this invention to provide a methodof producing a vaccine which is an attenuated or avirulent pathogenover-expressing a homologous antigen, and immunizing an animal,including humans, with the vaccine such that the vaccine induces astrong cell mediated or antibody mediated immune response against avirulent pathogen, thereby providing complete protection, such assterile immunity, against a challenge by the virulent pathogen.

It is a further object of the invention to provide a method of producinga vaccine which is an attenuated or avirulent pathogen over-expressing ahomologous antigen, and immunizing an animal with the vaccine such thatthe vaccine causes over-expression of an homologous antigen andexpression of a heterologous antigen, both of which provide protectionagainst the virulent pathogen in the vaccinated subject.

It is yet a further object of this invention to provide anover-expressing homologous vaccine, a means for making such a vaccineand a method of using the vaccine for prophylaxis and treatment ofBrucellosis in animals, especially bovine animals.

BRIEF DESCRIPTION OF THE DRAWINGS

The attached figures are intended to aid in explaining and to moreparticularly point out the invention described herein. In particular:

FIG. 1 is a diagram depicting the derivation of a homologous antigenfrom a Brucella species, and insertion of the antigen into a Brucellaspecies vaccine strain;

FIGS. 2A and 2B depicts construction of recombinant plasmids forover-expression of copper/zinc SOD(A) and GroES and GroEL(B) in B.abortus strain RB51;

FIG. 3 demonstrates the clearance of B. abortus strain 2308 from thespleens of mice vaccinated with B. abortus strain RB51 over-expressingcopper/zinc SOD or GroES/EL; and

FIG. 4 demonstrates the cytotoxic activity by lymphocytes towardBrucella infected cells from mice vaccinated with B. abortus strain RB51over-expressing copper/zinc SOD or GroES/EL.

DETAILED DESCRIPTION

The invention is directed to a vaccine for the immunization ofvertebrates against disease caused by a pathogen, wherein the vaccinecomprises an attenuated or avirulent pathogen that over-expresses one ormore homologous antigens encoded by at least one gene from the pathogen,wherein each antigen is capable of inducing a protective immune responseagainst the pathogen.

This over-expressing homologous antigen vaccine is produced by geneticengineering of live, attenuated microbes by a process having the stepsof: a) selecting a gene encoding an homologous antigen capable ofdirectly or indirectly stimulating protective immunity against apathogenic micro-organism (pathogen), and b) inserting said gene into anattenuated or avirulent version of the pathogen such that the homologousantigen is over-expressed. The resultant over-expressing homologousantigen vaccine (OHAV) is more specifically prepared by the followingsteps:

a) extracting deoxyribonucleic acid from a pathogenic micro-organism;

b) identifying a gene from the deoxyribonucleic acid, wherein said geneencodes at least one antigen capable of stimulating protective immunityagainst the pathogenic micro-organism;

c) inserting said gene into a plasmid capable of replication andexpression in the pathogenic micro-organism; and

d) introducing said plasmid into an attenuated or avirulent version ofthe pathogenic micro-organism.

The resultant vaccine synthesizes the antigen as a result oftranscription and translation of the gene located in at least two sites,i.e., the genome and the plasmid. In particular, it is preferred thatthe plasmid be a multicopy type, so that it may produce a greater numberof the protective antigen than the single genomic copy otherwisegenerated.

The above method may be used to create over-expressing homologousantigen vaccines for many different diseases. The over-expression of theantigen usually increases both the T cell and antibody immune response,thereby greatly increasing the level of protection in the subject.Because both types of immune response are improved, both intracellularand extracellular pathogens are affected, thereby providing greaterprotection against the pathogen.

For example, a vaccine against the pathogenic micro-organism Brucellamay be produced. In particular, the pathogen may be selected from anyspecies of Brucella, including B. abortus, B. melitensis, B. ovis, B.suis, B. canis and B. neotomae. The pathogen used to produce the vaccineis preferably selected from a specific strain of Brucella, such as B.abortus strain 19, B. abortus strain RB51, B. melitensis strain VTRM1,B. suis strain VTRS1 and B. melitensis strain REV1.

It is particularly advantageous that the vaccine be prepared with one ormore of a Cu/Zn SOD gene, a GroES gene or a GroEL gene of B. abortusstrain RB51. In particular, it is preferred that the above genes beobtained from a pUC19 genomic library of B. abortus strain 2308.

A vaccine produced according to the above specifications is particularlyeffective for prophylaxis or treatment of diseases such as Brucellosis.For example, an effective vaccine for prophylaxis or treatment of abovine animal against Brucellosis according to the invention is anattenuated or avirulent derivative of B. abortus strain RB51 capable ofover-expressing at least one homologous antigen. In particular, it ispreferred that the antigen be encoded by one or more of a Cu/Zn SODgene, a GroES gene or a GroEL gene, preferably selected from a pUC19genomic library of B. abortus strain 2308. It is even more preferablethat the attenuated or avirulent derivative also express a heterologousantigen capable of inducing protective immunity against B. abortus.

The method of prophylaxis or treatment of a vertebrate suffering from apathogenic micro-organism is as follows:

a) extract deoxyribonucleic acid from the pathogenic micro-organism;

b) identify at least one gene encoding at least one antigen from thedeoxyribonucleic acid, wherein the antigen is capable of stimulatingprotective immunity against the pathogenic micro-organism;

c) insert the at least one gene into a plasmid capable of replicatingand expressing in the pathogenic micro-organism;

d) transform an attenuated or avirulent version of the pathogenicmicro-organism with the plasmid to form a vaccine; and

e) administer an effective amount of the vaccine to the vertebrate.

The vaccine used for the method for prophylaxis and treatment may be anoriginal vaccine strain or a modified existing vaccine strain. Forexample, B. abortus strain RB51 can be modified to over-express ahomologous antigen, thereby producing a new strain capable of use in avaccine for the prophylaxis or treatment of Brucellosis, particularly inbovine animals.

In particular, a new Brucella vaccine can be prepared by: 1) selecting agene encoding a protective antigen from a strain of Brucella; 2)inserting the gene from the pathogen into a multicopy plasmid capable ofreplication and expression in Brucella; and 3) introducing the plasmidinto Brucella by means such as transformation. One or more homologousantigens may be over-expressed in this manner. Additionally, one or moreheterologous antigens may be expressed in the vaccine by methods knownin the art.

By over-expressing one or more homologous antigens of a given pathogen,greater T cell and/or antibody immune response against that pathogen isstimulated in the vertebrate treated with the vaccine produced from theattenuated or avirulent pathogen, affording greater protection againstthe unattenuated pathogen. Further protection may be offered byadditional expression of one or more heterologous antigens by theattenuated or avirulent pathogen by means known to one of ordinary skillin the art.

The resultant over-expressing homologous antigen vaccine may beadministered in a dose effective to promote prophylaxis or treatment ofa disease caused by the pathogen in the desired subject vertebrate. Asknown to one of ordinary skill in the art, dosages should be adjustedfor each subject based on factors such as weight, age, and environmentalfactors. The effective dose may be administered in any effective mannerbased on the type of animal being treated, its age and condition.

EXAMPLES Example 1

Two OHAVs were constructed by over-expressing either the Cu/Zn SOD geneor the GroES and GroEL genes in B. abortus strain RB51. The genes forCu/Zn SOD, GroES and GroEL were initially obtained from a pUC19 genomiclibrary of B. abortus strain 2308. As shown in FIG. 2, the insertscontaining these genes along with their own promoters were excised fromthe pBA113 (SOD) and pBA2131 (GroES and GroEL) regions and subclonedinto pBBR1MCS, a broad-host range plasmid which has routinely been usedin Brucella research. The resulting recombinant plasmids were termed aspBBSOD and pBBGroES/EL (FIG. 2). The B. abortus strain RB51 wastransformed with these plasmids by electroporation. Brucella containingthe plasmids were selected by plating the transformed bacteria ontrypticase soy agar plates containing 30 μg/mL of chloramphenicol. Todetermine the over-expression of the cloned genes, the antibioticresistant colonies were individually grown in trypticase soya broth andthe bacterial extracts used as antigens in an immunoblot analysis.Strain RB51 containing pBBSOD (RB51SOD) and pBBGroES/EL (RB51GroESL)over-expressed Cu/Zn SOD and GroEL, respectively, as compared to strainRB51 containing pBBR1MCS alone (RB51pBB).

Protection studies in mice:

Groups of 8 mice were vaccinated by inoculating, intraperitoneally,4×10⁸ colony forming units (cfu) of either strain RB51SOD, RB51GroESL,RB51pBB or RB51 in 0.5 mL of saline. One group of mice was inoculatedwith 0.5 mL of saline as a control. After 6 weeks, 5 mice in each groupwere challenged intraperitoneally with 2.5×10⁴ cfu of virulent strain2308. The remaining three mice in each group were used to characterizethe immune responses. Two weeks after challenge with virulent strain2308, mice were euthanized and the cfu of strain 2308 per spleen weredetermined. Mice immunized with strain RB51SOD had a significantly lowernumber of bacteria as compared to those immunized with strain RB51. Inmice immunized with strain RB51GroESL, the number of bacteria observablewas at the lower limit (<20 cfu/spleen) of the detection method.

Characterization of immune responses

After 6 weeks of vaccination, serum was collected from 3 mice in eachgroup for analysis of the humoral antibody response. These mice wereeuthanized and the lymphocytes harvested from their spleens were used tostudy the cell-mediated immune response. As shown in FIG. 3, micevaccinated with strain RB51 developed antibodies to GroEL but did notdevelop antibodies to Cu/Zn SOD. In contrast, mice vaccinated withstrain RB51SOD developed a strong antibody response to Cu/Zn SOD, andmice vaccinated with strain RB51GroESL developed a stronger antibodyresponse to GroEL protein (FIG. 3) than that exhibited by strain RB51vaccinated mice. These results indicate an enhanced antibody response bythe OHAV.

The cell mediated immune response caused was characterized bydetermining the cytotoxic activity of lymphocytes toward Brucellainfected cells. Specific splenic lymphocyte activity was enhanced invitro by co-culturing with mitomycin C treated Brucella infectedmacrophages as stimulator cells. A cytotoxicity assay was performedusing enhanced lymphocytes as effector cells (E) and Brucella infectedmacrophages as target cells (T). In the assay, E and T cells were mixedin two different ratios, 10:1 and 5:1. The percent specific lysis oftarget cells was calculated for each E:T ratio using standard methods(FIG. 4). Lymphocytes from mice vaccinated with RB51SOD or RB51GroESLshowed enhanced cytotoxic activity relative to saline or strain RB51vaccinated mice. This increased cytotoxic lymphocyte activity (indicatedby the increased % specific lysis) directly correlates with the observedenhanced protection of mice against challenge with virulent B. abortusstrain 2308; the higher the protective level, the higher the specificcytotoxic activity.

Example 2

An OHAV is constructed by over-expressing the ctxB gene in Vibriocholera. The gene is obtained from the deoxyribonucleic acid of thepathogen and inserted into a plasmid capable of replicating andexpressing in the pathogen. The resulting recombinant plasmid is used totransform Vibrio cholera by means of electroporation. Plasmids areplated and selected by means known in the art. The resultantover-expressing homologous antigen vaccine strain promotesoverproduction of antibodies that neutralize the cholera toxin, therebyproviding greater protection for prophylaxis and treatment of cholera inhumans.

Example 3

An OHAV is constructed by over-expressing the groEL gene ofMycobacterium tuberculosis in a Mycobacterium species. The gene isobtained from the deoxyribonucleic acid of the pathogen and insertedinto a plasmid capable of replicating and expressing in the pathogen.The resulting recombinant plasmid is used to transform a Mycobacteriumspecies by means of electroporation. Plasmids are plated and selected bymeans known in the art. The resultant over-expressing homologous antigenvaccine strain promotes overproduction of GroEL proteins, therebyproviding greater protection for prophylaxis and treatment oftuberculosis in humans. In particular, over-expression of the groEL geneencoding the GroEL protein in M. bovis BCG provides greater protectiveimmunity against tuberculosis because BCG vaccines are known to targetantigen protecting cells, such as macrophages, thereby providing a meansof introducing the antigens into the T cells, inducing protective cellmediated immunity.

The above examples are illustrative only. The scope of the invention isnot limited to the examples, but is described in the specification andaccompanying claims. Those of ordinary skill in the art will recognizemethods and materials which could be substituted for those describedabove, and any such methods and materials are intended to be covered bythe above disclosure and following claims.

What is claimed is:
 1. A vaccine for immunization, prophylaxis ortreatment of a vertebrate at risk of or suffering from Brucellosis,wherein said vaccine comprises an attenuated or avirulent strain of anotherwise pathogenic bacteria of the genus Brucella, and wherein saidstrain over-expresses at least one homologous antigen encoded by atleast one gene from said bacteria and wherein said at least one antigenis capable of inducing a protective or therapeutic immune response inthe vertebrate against Brucellosis.
 2. The vaccine of claim 1, whereinsaid attenuated or avirulent strain of said bacteria further expressesone or more heterologous antigens from at least one other pathogen, andwherein said heterologous antigen is capable of inducing a protective ortherapeutic immune response in the vertebrate against said otherpathogen.
 3. The vaccine of claim 1, wherein the bacteria is selectedfrom the group consisting of B. abortus, B. melitensis, B. suis, and B.canis.
 4. The vaccine of claim 1, wherein the bacteria is B. abortus. 5.The vaccine of claim 4, wherein the at least one gene is a Cu/Zn SODgene.
 6. The vaccine of claim 5, wherein the Cu/Zn SOD gene is obtainedfrom a pUC19 genomic library of B. abortus strain
 2308. 7. The vaccineof claim 4, wherein the at least one gene is one or both of a GroES geneand a GroEL gene.
 8. The vaccine of claim 7, wherein the GroES gene andthe GroEL gene are obtained from a pUC19 genomic library of B. abortusstrain
 2308. 9. The vaccine of claim 1, wherein the vertebrate isbovine.
 10. An attenuated or avirulent strain of B. abortus thatover-expresses at least one homologous antigen encoded by at least onegene from said B. abortus, and wherein said at least one antigen iscapable of stimulating a protective or therapeutic immune responseagainst Brucellosis.
 11. The attenuated or avirulent strain of B.abortus of claim 10, wherein the at least one homologous antigen isencoded by at least one gene selected from the group consisting of aCu/Zn SOD gene, a GroES gene and a GroEL gene.
 12. A method forimmunization, prophylaxis or treatment of a vertebrate at risk of orsuffering from Brucellosis comprising administering an effective amountof a vaccine, wherein said vaccine comprises an attenuated or avirulentstrain of an otherwise pathogenic bacteria of the genus Brucella thatover-expresses at least one homologous antigen encoded by at least onegene from said bacteria and wherein said at least one antigen is capableof inducing a protective or therapeutic immune response in thevertebrate against Brucellosis.
 13. The method of claim 12, wherein saidattenuated or avirulent strain further expresses one or moreheterologous antigen from at least one other pathogen, and wherein saidheterologous antigen is capable of inducing a protective or therapeuticimmune response in the vertebrate against said other pathogen.
 14. Themethod of claim 12, wherein the at least one gene is a Cu/Zn SOD gene inB. abortus strain RB51.
 15. The method of claim 14, wherein the Cu/ZnSOD gene is obtained from a pUC19 genomic library of B. abortus strain2308.
 16. The method of claim 12, wherein the at least one gene is oneor both of a GroES gene and a GroEL gene in B. abortus strain RB51. 17.The method of claim 16, wherein the GroES gene and the GroEL gene areobtained from a pUC19 genomic library of B. abortus strain
 2308. 18. Thevaccine of claim 4, wherein the attenuated or avirulent strain of saidbacteria is B. abortus strain RB51.
 19. The attenuated or avirulentstrain of B. abortus of claim 10, wherein the attenuated or avirulentstrain of B. abortus further expresses one or more heterologous antigensfrom at least one other pathogen, and wherein said heterologous antigenis capable of inducing a protective or therapeutic immune responseagainst said other pathogen.
 20. The attenuated or avirulent strain ofB. abortus of claim 10, wherein the attenuated or avirulent strain of B.abortus is B. abortus strain RB51.
 21. The method of claim 12, whereinsaid pathogenic bacteria is B. abortus.
 22. The method of claim 21,wherein said attenuated or avirulent strain of said pathogenic bacteriais B. abortus strain RB51.
 23. The method of claim 12, wherein the atleast one homologous antigen is encoded by at least one gene selectedfrom the group consisting of a Cu/Zn SOD gene, a GroES gene and a GroELgene.